IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression - PubMed (original) (raw)
IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression
Kathrin Eller et al. J Immunol. 2011.
Abstract
Both mast cells (MCs) and regulatory T cells (Tregs) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MCs and Tregs have been shown to play a protective role. Transfer of wild-type (wt) Tregs into wt recipients almost completely prevents development of NTS and leads to a profound increase of MCs in the renal draining lymph nodes (LNs). By contrast, transfer of wt Tregs into animals deficient in MCs, which are characterized by an exaggerated susceptibility to NTS, no longer exhibited protective effects. Blocking the pleiotropic cytokine IL-9, known to be involved in MC recruitment and proliferation, by means of a mAb in mice receiving Tregs abrogated protection from NTS. Moreover, transfer of IL-9-deficient Tregs also failed to protect from NTS. In the absence of Treg-derived IL-9, MCs fail to accumulate in the LNs, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Tregs. In summary, to our knowledge, we provide the first direct in vivo evidence that the nephroprotective, anti-inflammatory effects of Tregs critically depend on IL-9-mediated attraction of MCs into kidney-draining LNs.
Figures
Figure 1. Treg transfer increases MC in kidney-draining LN
Kidney-draining LN of animals receiving either Treg (white bar) or control T cells (black bar; n=13 per group) were analyzed for MC infiltration fourteen days after disease induction. (A) Real-time PCR for the expression of MC-tryptase is shown. Data are presented as x-fold increase as compared to LN of healthy controls. (B) Giemsa staining of LN. The number of MC per mm2 is given. The data are expressed as mean ± SEM. *p<0.05. (C) A representative example of a Giemsa-stained LN section from a Treg-injected mouse 14 days after induction of NTS is shown. MC are marked by arrows. Magnification x400.
Figure 2. Treg in MC-deficient KitW/KitW-v and Kit+/+ mice
Fourteen days after NTS was induced in KitW/KitW-v (white bar) and Kit+/+ mice (black bar; n=10 per group), LN were evaluated for Treg infiltration by (A) performing real-time PCR for the detection of FoxP3 and by (B) flow cytometric analysis for CD4+CD25+FoxP3+ cells. The real-time data are expressed as x-fold increase of FoxP3/beta-actin compared to mRNA isolated from healthy control LN (which was set as 1). (C and D) Evaluation of the immune-suppressive potential of CD4+CD25+ Treg from healthy (C) Kit+/+ mice and (D) MC-deficient KitW/KitW-v. Treg from Kit+/+ or MC-deficient KitW/KitW-v animals were co-incubated with CD4+CD25− Kit+/+ cells in a 1:1 and 1:5 ratio. Proliferation was measured by 3H-thymidine incorporation after 7 days and the percentage of proliferation compared to the respective control CD4+CD25− T cell population is shown. Moreover, both strains were subjected to Treg quantification, as shown by representative FACS-stainings shown in the inserts in C and D.
Figure 3. MC-deficient animals with NTS are resistant to the anti-inflammatory Treg effects
KitW/KitW-v (grey and light grey bar) and Kit+/+ mice (black and white bar) received either 5×106 Treg (light grey and white bar) or control T cells (grey and black bar) and were subjected to NTS (n=8 per group). (A) 7 and 14 days after induction of NTS albumin and creatinine in the urine were evaluated. The urinary albumin/creatinine ratio is given in mg/mg. (B) Representative PAS staining from tissue sections of kidneys from the indicated groups 14 days after induction of NTS are shown. Magnification x400. KitW/KitW-v receiving either Treg or control T cells display crescent formation as highlighted by black arrows. Only few crescent formations were seen in Kit+/+ control mice, but they were found to have, as KitW/KitW-v mice, PAS-positive deposits in their glomeruli (black asterix) as well as infiltrating inflammatory cells. When Kit+/+ mice were treated with Treg no glomerular pathologies were detected. (C) Semiquantitative analysis of PAS deposits in kidney sections according to the PAS-score described in the Material and Methods section is shown. (D) T cell infiltration pattern in the indicated groups are shown by semiquantitative quantification of CD4 and CD8 stained tissue sections. The number of positive cells in 6 HPF is given. (E-F) Kidney infiltration patterns of (E) F4/80+ or (F) CD68+ cells, which were stained and counted according to the F4/80 and CD68-scoring procedure described in the Material and Methods section. (G) Rabbit anti-mouse titers were evaluated in the serum of respective mice 14 days after induction of NTS. All data are presented as mean ± SEM. * … provides the significance between Kit+/+ mice receiving either Treg or control cells (p<0.025). # … provides the significance between KitW/KitW-v and Kit+/+ mice receiving control cells (p<0.025). Three independent experiments were performed.
Figure 4. Increased inflammation in MC-deficient animals suffering from NTS
NTS was induced in KitW/KitW-v and Kit+/+ mice (n=6 per group). Seven days after induction of NTS the mRNA expression of IFN-γ, IL-6, IL-17A, IL-10, Gata-3 and TGF-β was evaluated in the LN. The real-time PCR data are expressed as x-fold increase of the respective parameter/beta-actin compared to the mean expression of Kit+/+ LN (which was set as 1). All data are presented as mean ± SEM. *p<0.05.
Figure 5. Systemic IL-9 blockade prevents nephroprotection by Treg
NTS animals received either Treg (white bar; n=5) or control T cells (black bar; n=5) in combination with an isotype antibody control. A third group was injected with Treg in combination with an anti-IL-9 blocking mAb, which was applied every second day (grey bar; n=5). (A) The albumin/creatinine ratio in the urine was evaluated 7 and 14 days after induction of NTS. (B) PAS stained sections were counted according to the PAS-score as described in the Material and Methods section. (C) Sections were stained with Giemsa and the number of MC per mm2 was evaluated. (D) The kidney-draining LN was evaluated for MC infiltration by real-time PCR for MC tryptase 1 mRNA. The data are expressed as x-fold increase of MC tryptase 1/beta-actin compared to mRNA isolated from healthy control LN (which was set as 1). All data are presented as mean ± SEM. *p<0.025. Two independent experiments were performed.
Figure 6. Treg-derived IL-9 is critical for MC-dependent nephroprotection in NTS
NTS mice received either wt-Treg (white bar), control T cells (black bar) or Treg isolated from IL-9 deficient animals (grey bar). (A) The albumin/creatinine ratio in the urine was evaluated 7 (n=7-9) and 14 days (n=5-7) after induction of NTS. (B) Representative PAS stained kidney sections from the indicated groups 14 days after induction of NTS are shown. Magnification x400. Mice receiving either control cells or IL-9 deficient Treg displayed crescent formations (black arrows) and large PAS-positive deposits (black asterix) in their glomeruli, whereas Treg treated mice were found to have only marginal PAS-positive deposits and no crescent formations. (C) Quantification of PAS deposition by the PAS-score is given for the respective groups. (D) The kidney-draining LN was evaluated for MC infiltration by real-time PCR for MC tryptase 1 mRNA. The data are expressed as x-fold increase of MC tryptase 1/beta-actin compared to healthy control LN (which was set as 1). All data are presented as mean ± SEM. *p<0.05. (E) Renal cryosections of the three groups were stained for fibrin deposition. No significant differences were observed. Representative pictures are shown. Magnification x400. (F) Evaluation of the immune-suppressive potential of CD4+CD25+ Treg from IL-9 deficient animals. Treg from wt or IL-9 deficient animals were co-incubated with CD4+CD25− wt cells in a 1:1 ratio. Proliferation was measured by 3H-thymidine incorporation after 7 days and the percentage of proliferation compared to the respective control CD4+CD25− T cell population is shown. The data are presented as mean ± SEM; *p<0.025; n.s. not significant. Two independent experiments were performed.
Figure 7. Regulation of the chemokines Cxcl-1, -2, -5 in the kidney draining LN
(A) NTS mice received either wt Treg (white bar), control T cells (black bar) or Treg isolated from IL-9 deficient animals (grey bar). (B) NTS animals received either Treg (white bar; n=5) or control T cells (black bar; n=5) in combination with an isotype antibody control. A third group received Treg in combination with an anti-IL-9 blocking mAb, which was applied every second day (grey bar; n=5). (A and B) In both experiments, the kidney-draining LN was evaluated for the chemokines Cxcl-1, -2, -5 by real-time PCR. The data are expressed as x-fold increase of the respective chemokine/beta-actin ration compared to healthy control LN (which was set as 1). All data represent the mean value ± SEM. *p<0.025.
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